Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection primarily depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of Y. pestis parent and mutant strains. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. We have also established in vitro methods to infect fleas and to monitor transmission dynamics of fleas over a one-month period following their infectious blood meal. With this system we are able to compare the relative importance of the two modes of transmission and the relative vector competence of different flea species. Our studies of flea vector competence and vectorial capacity will be useful to develop more realistic mathematical modeling of the epidemiology of plague transmission and the conditions that lead to plague epizootics. During FY2017, we published a study on the comparative vector competence of the North American ground squirrel flea O. montana and the rat flea X. cheopis. The work described new protocols to monitor and compare the infection and transmission dynamics of different flea vector species following a single standardized infectious blood meal. Several recent papers cite that O. montana rarely if ever transmits by the biofilm-dependent mechanism, whereas X. cheopis is the vector par excellence by this mechanism. However, we demonstrated that both fleas are proficient at this transmission mechanism, thereby correcting a misconception. Another aspect of this study was to compare the kinetics and temporal patterns of transmission during the early-phase and later phase transmission windows. We found that the later, biofilm-dependent transmission mechanism was much more efficient than early-phase transmission. We also published evidence for our proposed model for the early-phase transmission mechanism. Early-phase transmission was long assumed to be mechanical, via contaminated mouthparts. However, we proposed a regurgitative model based on the striking finding that dense bacterial aggregates associated with a rough, amorphous, brown-colored, waxy-looking material localized to the foregut within a few hours after an infectious blood meal. In some fleas, these bacterial aggregates could obstruct blood flow during the fleas next feeding attempt, which correlated with transmission. Thus, we proposed a unified theory of flea-borne transmission in which both early and later transmission modes occur by regurgitation from fleas in which the foregut is obstructed by masses of Y. pestis at different stages of biofilm maturation. We refined and evaluated new experimental systems to maintain and monitor infection status and transmission efficiency of individual fleas at different times after infection. The data will be used to estimate values for important parameters such as the probability of flea vectors developing a transmissible infection after feeding on a bacteremic host and the transmission efficiency during a four-week period after infection. Limited data are available are currently available for these values, which are needed for understanding plague epidemiology. In collaboration with Dr. Angela Luis at the University of Montana, we have developed mathematical models to better understand conditions that give rise to periodic plague epizootics, and will use our experimentally derived data in these models. In 2017 we published a summary of many of our experimental protocols for infecting and monitoring fleas on the BEI Resources website (https://www.beiresources.org/Catalog/VectorResources.aspx) as part of their program to provide arthropod vector resources widely available to the research community. We established a collaboration with USGS and USFWS biologists working on a sylvatic plague vaccine for prairie dogs in Montana and South Dakota. Prairie dog colonies are subject to periodic explosive plague epizootics that can essentially extirpate the colony, which pose a public danger to rural communities and hinder efforts to reintroduce the black-footed ferret, an endangered species. The conditions that give rise to prairie dog epizootics are enigmatic. Based on one limited 1940 study, the predominant flea of prairie dogs, O. hirsuta, is a poor vector. Because of this, alternate transmission routes in addition to the classic flea-borne route have been proposed, none of which add up. We will use our flea infection and transmission monitoring experimental systems to reevaluate the vector competence of prairie dog fleas, which will be collected and provided to us by our USGS and USFWS collaborators. Similarly, Dr. James Belthoff, Boise State University, is providing P. irritans fleas, which we will also evaluate. This flea is thought to be a poor vector but has controversially been hypothesized to have transmitted Y. pestis from human to human during the European plague epidemics of the Middle Ages. Reliable vector competence data regarding these fleas will enable more realistic modeling of these epizootiologic/epidemiologic scenarios. In collaboration with the Genomics Unit of the RML Research Technologies Branch, we are examining and characterizing the transcriptomes of Y. pestis, Y. pseudotuberculosis, and our biofilm-producing Y. pseudotuberculosis mutant during growth in vitro and during infection of the flea, with the goal of identifying genes and gene regulatory pathways that are important for flea-borne transmission. The in vivo and in vitro transcriptomic comparisons between these three strains are designed to broadly identify candidate components of biofilm regulatory pathways and other genes important for the recent evolutionary adaptation to flea-borne transmission. Significant differences in the expression of orthologous genes in the flea might be indicative of evolutionary changes in gene regulatory pathways. In a second collaboration with the Genomics Unit, the gene expression response of the flea digestive tract epithelial cells to sterile and infected blood meals was characterized and compared to that of unfed fleas. Data analysis will disclose the flea digestive system pathways and immune response to oral infection with Y. pestis, about which nothing is known.